Dynamic epigenetic alteration is central to differentiation of mammalian sperm, however the nature of these changes remains largely unknown. We are using the process of sporulation in the budding yeast S. cerevisiae, as a tractable model for mammalian spermatogenesis, to uncover dynamic chromatin and epigenetic regulation of transcription, meiosis and chromatin compaction. Our previous observations indicate that there are dramatic temporal changes in chromatin during sporulation, including histone modifications and other alterations. Further, our results indicate that mouse sperm differentiation involves similar temporal sequences of histone modifications, which are analogous in timing to the yeast. This conservation of the pattern of histone modifications during gametogenesis from yeast to mammals, strongly indicates that epigenetic regulation is fundamental to the normal process of chromatin restructuring during gametogenesis. Our hypothesis is that chromatin modulation is a highly evolutionarily conserved process in gametogenesis, and thus is a key regulatory feature of both yeast sporulation and mammalian spermatogenesis. We will address chromatin mechanisms during gametogenesis, through the following specific aims: (1) investigate chromatin mechanisms that operate through histones H3 and H4, including novel post-translational modifications and other regulatory features, identified via mutational screening in the previous funding period, (2) complete screening for histone substitution mutations in histone H2A and H2B that decrease or increase sporulation, and unravel their mechanisms through post-translational modifications, and other regulatory mechanisms, (3) examine linker histone Hho1-mediated mechanisms involved in meiotic gene transcriptional repression and post-meiotic chromatin compaction. As an important part of our proposal, we will determine whether these novel chromatin alterations are conserved during mouse spermatogenesis. Overall, results from these studies will provide novel views of dynamic changes in chromatin structure and function. PUBLIC HEALTH RELEVANCE: We previously discovered novel epigenetic modifications during mouse spermatogenesis, starting from a model system in a simple organism, yeast. In this proposal, we will discover additional epigenetic changes in yeast, and determine whether they are conserved in the mouse process. Results from this study will inform understanding of epigenetic regulation of normal human spermatogenesis and dysfunction in male infertility.